Myoferlin depletion elevates focal adhesion kinase and paxillin phosphorylation and enhances cell-matrix adhesion in breast cancer cells.

Breast cancer is the second leading cause of malignant death among women. A crucial feature of metastatic cancers is their propensity to lose adhesion to the underlying basement membrane as they transition to a motile phenotype and invade surrounding tissue. Attachment to the extracellular matrix is mediated by a complex of adhesion proteins, including integrins, signaling molecules, actin and actin-binding proteins, and scaffolding proteins. Focal adhesion kinase (FAK) is pivotal for the organization of focal contacts and maturation into focal adhesions, and disruption of this process is a hallmark of early cancer invasive potential. Our recent work has revealed that myoferlin (MYOF) mediates breast tumor cell motility and invasive phenotype. In this study we demonstrate that noninvasive breast cancer cell lines exhibit increased cell-substrate adhesion and that silencing of MYOF using RNAi in the highly invasive human breast cancer cell line MDA-MB-231 also enhances cell-substrate adhesion. In addition, we detected elevated tyrosine phosphorylation of FAK (FAK(Y397)) and paxillin (PAX(Y118)), markers of focal adhesion protein activation. Morphometric analysis of PAX expression revealed that RNAi-mediated depletion of MYOF resulted in larger, more elongated focal adhesions, in contrast to cells transduced with a control virus (MDA-231(LVC) cells), which exhibited smaller focal contacts. Finally, MYOF silencing in MDA-MB-231 cells exhibited a more elaborate ventral cytoskeletal structure near focal adhesions, typified by pronounced actin stress fibers. These data support the hypothesis that MYOF regulates cell adhesions and cell-substrate adhesion strength and may account for the high degree of motility in invasive breast cancer cells.

Exocyst complex protein expression in the human placenta.

INTRODUCTION: Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. OBJECTIVE: While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. METHODS: A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. RESULTS: The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion. DISCUSSION/CONCLUSION: Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst's regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion.

IFPA Meeting 2013 Workshop Report II: use of 'omics' in understanding placental development, bioinformatics tools for gene expression analysis, planning and coordination of a placenta research network, placental imaging, evolutionary approaches to understanding pre-eclampsia.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At the IFPA meeting 2013 twelve themed workshops were presented, five of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of new technologies for placenta research: 1) use of 'omics' in understanding placental development and pathologies; 2) bioinformatics and use of omics technologies; 3) planning and coordination of a placenta research network; 4) clinical imaging and pathological outcomes; 5) placental evolution.

IFPA Meeting 2012 Workshop Report I: comparative placentation and animal models, advanced techniques in placental histopathology, human pluripotent stem cells as a model for trophoblast differentiation.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2012 there were twelve themed workshops, three of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of models and technical issues involved in placenta research: 1) comparative placentation and animal models; 2) advanced techniques in placental histopathology; 3) human pluripotent stem cells as a model for trophoblast differentiation.

A placental sub-proteome: the apical plasma membrane of the syncytiotrophoblast.

As a highly vascularized tissue, the placenta mediates gas and solute exchange between maternal and fetal circulations. In the human placenta, the interface with maternal blood is a unique epithelial structure known as the syncytiotrophoblast. Previously we developed a colloidal-silica based method to generate highly enriched preparations of the apical plasma membrane of the syncytiotrophoblast. Using similar preparations, a proteomics assessment of this important sub-proteome has identified 340 proteins as part of this apical membrane fraction. The expression of 38 of these proteins was previously unknown in the human placental syncytiotrophoblast. Together with previous studies, the current proteomic database expands our knowledge of the proteome of the syncytiotrophoblast apical plasma membrane from normal placentas to include more than 500 proteins. This database is a valuable resource for future comparisons to diseased placentas. Additionally, this data set provides a basis for further experimental studies of placenta and trophoblast function.

IFPA Meeting 2011 workshop report III: Placental immunology; epigenetic and microRNA-dependent gene regulation; comparative placentation; trophoblast differentiation; stem cells.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.

Placental dysferlin expression is reduced in severe preeclampsia.

Dysferlin (DYSF) and myoferlin (MYOF), members of the ferlin family of membrane proteins, are co-expressed in human placental syncytiotrophoblast (STB). Although the role of these ferlin proteins in the placenta has yet to be established, it has been suggested that DYSF and MYOF may contribute to the stability of the apical STB plasma membrane. The release of STB-derived cellular debris increases in the setting of preeclampsia (PE), suggesting relative destabilization of the hemochorial interface. To test whether PE was associated with alterations in placental expression of DYSF and/or MYOF, a cross-sectional study was performed using specimens of villous placenta collected form women with severe PE (n=10) and normotensive controls (n=10). DYSF and MYOF expression were examined using quantitative real-time RT-PCR, immunoblotting, and immunofluorescence labeling of tissue specimens. Placental DYSF expression was 57% lower at the mRNA level (p=0.03) and 38% lower at the protein level (p=0.026) in severe PE as compared to normotensive subjects. There were no differences in placental MYOF protein or mRNA expression between these groups. No appreciable changes in the distribution of DYSF or MYOF within placental villi was observed in PE relative to control specimens. We conclude that DYSF expression is reduced in severe PE relative to gestational age-matched controls. As DYSF has a role in membrane repair, these data suggest a role for DYSF in the stability of the apical STB plasma membrane and may account, at least in part, for the increased shedding of microparticles from this membrane in PE.

Placental proteomics: a shortcut to biological insight.

Proteomics analysis of biological samples has the potential to identify novel protein expression patterns and/or changes in protein expression patterns in different developmental or disease states. An important component of successful proteomics research, at least in its present form, is to reduce the complexity of the sample if it is derived from cells or tissues. One method to simplify complex tissues is to focus on a specific, highly purified sub-proteome. Using this approach we have developed methods to prepare highly enriched fractions of the apical plasma membrane of the syncytiotrophoblast. Through proteomics analysis of this fraction we have identified over five hundred proteins several of which were previously not known to reside in the syncytiotrophoblast. Herein, we focus on two of these, dysferlin and myoferlin. These proteins, largely known from studies of skeletal muscle, may not have been found in the human placenta were it not for discovery-based proteomics analysis. This new knowledge, acquired through a discovery-driven approach, can now be applied for the generation of hypothesis-based experimentation. Thus discovery-based and hypothesis-based research are complimentary approaches that when coupled together can hasten scientific discoveries.

Proteomics of the human placenta: promises and realities.

Proteomics is an area of study that sets as its ultimate goal the global analysis of all of the proteins expressed in a biological system of interest. However, technical limitations currently hamper proteome-wide analyses of complex systems. In a more practical sense, a desired outcome of proteomics research is the translation of large protein data sets into formats that provide meaningful information regarding clinical conditions (e.g., biomarkers to serve as diagnostic and/or prognostic indicators of disease). Herein, we discuss placental proteomics by describing existing studies, pointing out their strengths and weaknesses. In so doing, we strive to inform investigators interested in this area of research about the current gap between hyperbolic promises and realities. Additionally, we discuss the utility of proteomics in discovery-based research, particularly as regards the capacity to unearth novel insights into placental biology. Importantly, when considering under studied systems such as the human placenta and diseases associated with abnormalities in placental function, proteomics can serve as a robust 'shortcut' to obtaining information unlikely to be garnered using traditional approaches.

Association of PAT proteins with lipid storage droplets in term fetal membranes.

As depots for neutral lipids, lipid storage droplets (LDs) accumulate with advancing gestation within the fetal membranes. Little is currently known about the proteins associated with the LDs of these cells. The PAT family [perilipin, adipose differentiation-related protein (ADRP), and tail-interacting protein of 47 kilodaltons (TIP47)] represents a unique group of proteins thought to contribute to LD formation and function. We examined the association of each of the PAT proteins with LDs of term fetal membranes. We found that large LDs of amnion epithelial cells were reactive for neutral lipid stains and simultaneously encoated with ADRP and TIP47, but not perilipin. Within the remaining cell types, LDs were frequently co-labeled with antibodies recognizing ADRP and TIP47; however, in cells harboring only small LDs, the majority of TIP47 labeling was cytoplasmic. Structures labeled with perilipin antibodies were present only in chorion laeve trophoblasts. Gene and protein expression analyses suggested this to be a small molecular weight perilipin isoform, such as that seen in steroidogenic cells. We conclude that LDs are heterogeneous among differing cell types of the fetal membranes. Subclassification of LDs based on associated proteins suggests that these organelles may serve specialized functions within individual cells.

In situ immunolabeling allows for detailed localization of prostaglandin synthesizing enzymes within amnion epithelium.

Detailed information regarding the subcellular distribution of proteins within amnion epithelial cells is a goal of numerous placental biologists. In this report, we describe a versatile technique for in situ immunolabeling in amnion that is as technically permissible as traditional immunolabeling of cultured cells and, when coupled with confocal laser scanning microscopy, is similarly capable of providing detailed information regarding subcellular protein distribution. Using antibodies directed against sequential enzymes of the prostaglandin E biosynthesis cascade, we compared this novel method with immunofluorescent labeling using amnion cells in primary culture and cryosections of reflected fetal membrane rolls. By several criteria, we observed morphological variation between the cells cultured in vitro and the tissue specimens. Despite general consistencies in immunostaining patterns between the cryosectioned specimens and those labeled in situ, morphological preservation was superior using the latter technique. Relative to the cryosectioned specimens, in situ immunostaining was advantageous in that it permitted improved sampling efficiency, and allowed regional variations in labeling to be observed in a more global context within the tissue. Our results demonstrate that in situ immunolabeling provides a useful adjunct or alternative to immunolabeling using membrane roll preparations.

Differentiation-dependent regulation of the cyclooxygenase cascade during adipogenesis suggests a complex role for prostaglandins.

AIM: A thorough understanding of the mechanisms of adipocyte differentiation and metabolism is important for the prevention and/or treatment of obesity and its complications, including type 2 diabetes mellitus. A complex role for prostaglandins (PGs) in adipogenesis is suggested. We examined the expression and cellular localization of enzymes in the cyclooxygenase (COX) cascade that synthesize PGs as well as the PG profile as a function of differentiation status in 3T3-L1 cells. METHODS: Murine 3T3-L1 preadipocytes were used as a model for studies of adipocyte differentiation induced by a hormone cocktail and compared with the parental fibroblastic line NIH 3T3. Both cell lines were incubated in maintenance medium or differentiation medium. Nine days after differentiation, the expression of enzymes in the COX cascade was evaluated by immunoblot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry, and PG formation was examined using enzyme immunoassay. RESULTS: A differentiation-dependent diminution of COX-1 and COX-2 mRNA and cognate proteins in 3T3-L1 cells was observed. PG release, including PGE(2), 6-keto PGF(1alpha), PGD(2) and 15d-PGJ(2), significantly decreased following differentiation in 3T3-L1 cells (anova/Tukey, p < 0.05). However, microsomal PGE synthase (mPGES) and lipocalin-type PGD synthase (L-PGDS) were selectively upregulated. Immunocytochemistry revealed that COX-1 and COX-2 became intracellularly more diffuse upon differentiation, whereas mPGES was redistributed to the nuclear compartment. CONCLUSIONS: Regulation of PG formation and COX-2 expression in 3T3-L1 cells is differentiation-dependent and involves changes in the levels of gene expression of the individual isoforms as well as redistribution of the enzymes within cellular compartments.

Topical application of acidic bupivacaine to the lumbar ganglion induces mechanical hyperalgesia in the rat.

UNLABELLED: To investigate the neurologic mechanisms of acidic local anesthetic-induced low back pain in humans, we administered bupivacaine and buffered saline at acidic or alkalinized pH at the L5 dorsal root ganglion (DRG) of rats via a hole drilled through the transverse process covering the DRG. Behavioral changes were tested before and after bupivacaine or saline administration. Results indicate that acute single-dose infusion of the DRG with bupivacaine (0.5%) at acidic pH (5.5) induced ipsilateral mechanical hyperalgesia that lasted for 7 days. Acute infusion of alkalinized bupivacaine (pH 7.2), however, caused only minor hyperalgesia that lasted <3 days. Similar results were obtained when bupivacaine was replaced with saline. Alternatively, chronic delivery of acidic saline to the DRG via a subcutaneously implanted osmotic pump resulted in a significant decrease in the withdrawal threshold on the ipsilateral hind paw that lasted for 10 days. In rats receiving chronic treatment of the DRG with alkalinized saline, mechanical hyperalgesia lasted for only 3 days. The results demonstrated that acidic bupivacaine deposited at the DRG causes pain and hyperalgesia when the effects of the local anesthetic have dissipated. These findings may explain the limited therapeutic effects of some acidic local anesthetics used for management of cancer-related and chronic back pain. IMPLICATIONS: Acidic bupivacaine administered at the L5 lumbar ganglion causes pain and hypersensitivity of the hind paw in the rat. These findings may explain the limited therapeutic effects of some acidic local anesthetics used for treatment of cancer-related and chronic back pain.

Physiologic effects of stellate ganglion block: a result of complete ganglion blockade or the vertical spread of local anesthetic?

Traditionally, stellate ganglion blockade has been used for the diagnosis and treatment of upper extremity sympathetic pain. However, this treatment has not been shown to provide adequate sympathetic blockade of the upper extremities. This study demonstrates that a carefully performed upper thoracic sympathetic block with imaging guidance can result in a successful sympathetic blockade of the upper extremities. This study furthermore demonstrates that the occurrence of a Horners syndrome is not a testimony to a successful sympathetic block of the upper extremities.

Multidisciplinary approach for the management of post-herpetic neuralgia in elderly patients.

Post-herpetic neuralgia is associated with significant distress and morbidity. The management of acute neuritis and/or post-herpetic neuralgia can be particularly difficult. A multidisciplinary approach is required. A team consisting of the primary care physician, pain specialist, neurologist, geriatrician, pain psychologist, psychiatrist, and a physiatrist with an integrated approach will provide the best results. Early interventional therapy with sympathetic nerve blocks may significantly decrease the need for long-term opioid therapy, as well as long-term use of anticonvulsants, antidepressants, or membrane stabilizers. Early referral to a multidisciplinary pain center may furthermore decrease the behavioral trauma and family disruption associated with this painful condition.

Phrenic nerve injury following scalenectomy in a patient with thoracic outlet obstruction.

We present a case in which a patient with normal pulmonary reserve experienced orthopnea and hypoxia secondary to unilateral diaphragmatic paralysis following right scalenectomy. This operation was performed in an attempt to relieve neurovascular compromise at the thoracic outlet. To our knowledge, this association has not been previously described in the literature.